Synthesis and antimalarial activity of N-benzylated (N-arylcarbamoyl)alkylphosphonic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2016.04.021

A series of novel and readily accessible N-benzylated (N-arylcarbamoyl)alkylphosphonate esters and related compounds have been prepared as potential antimalarial agents. Bioassays reveal that some of these compounds exhibit promising activity against Plasmodium falciparum, and exhibit no significant growth inhibition of HeLa cells.

Full text of DOI:10.1016/j.bmc.2016.04.021

Accepted Manuscript
Synthesis and Antimalarial Activity of N-Benzylated (N-Arylcarbamoyl)alkyl-
phosphonic Acid Derivatives
Christiana M. Adeyemi, Faridoon, Michelle Isaacs, Dumisani Mnkandhla,
Heinrich C. Hoppe, Rui W.M. Krause, Perry T. Kaye
PII:
S0968-0896(16)30255-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.021
BMC 12936
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
1 February 2016
31 March 2016
9 April 2016
Please cite this article as: Adeyemi, C.M., Faridoon, Isaacs, M., Mnkandhla, D., Hoppe, H.C., Krause, R.W.M.,
Kaye, P.T., Synthesis and Antimalarial Activity of N-Benzylated (N-Arylcarbamoyl)alkylphosphonic Acid
Derivatives, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.021
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ArNH2
4a-e
O
O
O
O
Ar
Ar
N
POEt
N
P OH
OH
OEt
9
a-e
10c,d
GRAHICAL ABSTRACT
1

Synthesis and Antimalarial Activity of N-Benzylated
N-Arylcarbamoyl)alkylphosphonic Acid Derivatives
(
a
Christiana M. Adeyemi, Faridoon, Michelle Isaacs, Dumisani Mnkandhla,
a
c
c
b,c
Heinrich C. Hoppe, Rui W.M. Krause and Perry T. Kaye *
a,c
a,c
a
b
Department of Chemistry, Department of Biochemistry and Microbiolgy and Centre for Chemico- and
c
Biomedicinal Research, Rhodes University, Grahamstown, 6140, South Africa.
Abstract. A series of novel and readily accessible N-benzylated (N-arylcarbamoyl)-
alkylphosphonate esters and related compounds have been prepared as potential antimalarial
agents. Bioassays reveal that some of these compounds exhibit promising activity against
Plasmodium falciparum, and exhibit no significant growth inhibition of HeLa cells.
Keywords: Anti-malarial, N-benzylated phosphonate esters, PfDXR, aryl and heteroaryl derivatives
1. Introduction
The scourge of malaria continues to be a major health challenge in many countries, and there
are concerns that global warming might extend the habitat of infectious mosquito vectors into
1
regions which are, at present, malaria-free. The development of resistance to established
,2
antimalarial drugs has exacerbated the problem and necessitates the identification of new
3
drug targets and the development of new and effective drugs. 1-Deoxy-1-D-xylulose-5-
4,5
phosphate (DOXP; 1) reductoisomerase (PfDXR), is a critical enzyme in the non-
mevalonate, isoprenoid biosynthetic pathway in Plasmodium falciparum (Pf), it is parasite-
specific and there is considerable interest in developing effective inhibitors of this enzyme as
potential anti-malarial drugs.
OH
O
OH
N
O
OH
N
O
P
OH
H
P
OH
P
OH
O
OH
OH
OH
O
O
O
OH
1
2
3
_
_________
Corresponding author: Prof Perry Kaye. E-mail: P.Kaye@ru.ac.za
2

In our own research in this area, we have been exploring the synthesis and DXR-inhibition
potential of simplified and readily accessible analogues of fosmidomycin 2 − a naturally
6
,7
occurring DXR inhibitor and its acetyl derivative FR900098 3. Results from studies
involving N-aryl- and N-heteroarylcarboxamides have supported the importance of no more
than two methylene groups in the ‘hydrophobic patch’ between the metal chelating and
phosphonate moieties, and have indicated the presence of additional hydrophobic pockets
7
8
adjacent to the PfDXR active site. Kurz and co-workers have reported the DXR inhibition
potential of ‘reverse’ fosmidomycin analogues in which the carbonyl and hydroxylamine
moieties are transposed. In this communication, we now report the preparation and evaluation
of a series of N-benzylated ‘reverse’ fosmidomycin analogues, including N-benzylated
9
derivatives of compounds from our earlier studies which gave the best, albeit very modest,
EcDXR- and PfDXR-inhibition data. The key structural features of these analogues,
compared to fosmidomycin 2, are summarised in Figure 1 and include:- i) a common
phosphonate moiety as a replacement for the more labile phosphate group present in the
natural DXR substrate DOXP 1; ii) a bimethylene hydrophobic patch rather than the
trimethylene spacer in fosmidomycin 2; iii) an amide group as a reverse hydroxamate
replacement; and iv) a hydrophobic, N-benzyl group to occupy a hydrophobic pocket
adjacent to the DXR active site.
Hydroxamate
group
Hydrophobic
patch
(
a)
b)
Phosphonate
moiety
(
Amide moiety as
reverse hydroxamate
replacement
(
)n
Bulky
hydrophobic group
Figure 1. Structural similarities and differences between: (a) fosmidomycin 2; and
(b) scaffolds used in the present study.
3

2. Results and Discussion
2
.1. Chemistry
It was envisaged that the desired N-benzylated, bi-methylene-bridged phosphonate esters 9
(
or their mono-methylene-bridged analogues) could be accessed by direct benzylation
(
retrosynthetic option I, Scheme 1) of the amide nitrogen in the phosphonate esters 6 (or their
6,7
mono-methylene-bridged analogues 5), which had been synthesised previously (Scheme 2).
However, reactions of selected phosphonate esters with benzyl bromide in the presence of
1
base failed to afford the N-benzylated derivatives, and attention was turned to retrosynthetic
0
option II (Scheme 1). In this approach, N-benzylation is effected first.
O
O
Ar
N
H
P OEt
OEt
6
Option I
O
O
O
Ar
Ar
Ar
N
P
OEt
N
Cl
NH
OEt
ArNH2
Option II
9
8
7
4
Scheme 1. Retrosynthetic options to access the target molecules 9.
3
Thus, mixtures of each of the primary amines 4a-e, benzyl bromide (1 eq.) and NaHCO (1
eq.) in dry THF (Scheme 2) were stirred overnight at room temperature under an inert
atmosphere. Work-up and flash chromatography afforded the N-benzylated derivatives 7a-e
in yields of up to 72% (Table 1). In addition to the expected product 7b, the N,O-bis-
benzylated analogue, N-benzyl-3-[(benzyloxy)methyl]aniline 11 was also obtained as a new
but minor product (19%) from the reaction with 3-(hydroxymethyl)aniline 4b. Acylation of
the N-benzylated amines 7a-e was effected in THF using NaH, as base, and 3-
chloropropanoyl chloride. Work-up and flash-chromatography afforded the corresponding,
novel 3-chloropanamides 8a-e in low to moderate yield; the bis-acylated analogue 12 of the
phenolic derivative 8d was also obtained as a minor product (14%). Michaelis-Arbuzov
reactions of the 3-chloropanamides 8a-e in refluxing triethyl phosphite led to the series of
novel phosphonate esters 9a-e which were obtained as yellow oils in yields of up to 59%
4

(
Table 1) following flash- or preparative layer chromatography. The common method of
removing excess triethyl phosphite by washing with hexane was precluded by the solubility
of the products in hexane and, consequently, the crude products were purified
chromatographically. Given the design criteria discussed in the Introduction, attention was
focussed on the preparation of the bi-methylene-bridged, N-benzylated phosphonate esters 9.
O
ArNH2
a-e
i,ii
O
4
Ar
P OEt iii
OEt
N
H
n
X
iii
n = 0: 5a-e
n = 1: 6a-e
O
O
O
O
P
O
Ar
Ar
Ar
Ar
N
H
N
Cl
N
P OEt
OEt
N
OH
i
ii
iv
OH
For 9c,d
7a-e
8a-e
9a-e
10c,d
OH
Br
OH
NO2
ArNH =
2
NH2
NH2
N
NH2
NH2
NH2
a
b
c
d
e
Scheme 2.
Reagents and conditions:- (i) NaH, dry THF, then 3-chloropropanoyl chloride, r.t.; (ii)
triethyl phosphite, 6 h, 120-150 ºC; (iii) NaHCO , BnBr, dry THF; (iv) TMSBr, dry DCM,
, 24 h.
3
N
2
Table 1.
Percentage yields obtained for the N-benzylated derivatives 7a-e, the
-chloropopanamides 8a-e and the phosphonate esters 9a-e.
3
Ar
3
3
-Bromophenyl
-(Hydroxymethyl)phenyl
7a 65
7b 63
8a 50
8b 36
9a 51
9b 47
a
2
3
3
-Pyridyl
7c 56
7d 62
7e 72
8c 33
8d 39
8e 52
9c 49
9d 51
9e 59
b
-Hydroxyphenyl
-Nitrophenyl
a
b
Bis-benzylated analogue 11 (19%). Bis-2-chloropropanoyl analogue 12 (14%).
5

O
Si
O
O
O
HN
O
O
O
P
O
O
P
O
P
O
N
O
NH
NH
O
O
O
O
1
1
13
14
15
Additional compounds obtained in this study and evaluated for anti-malarial activity include:-
i) the phosphonate esters 13 and 14 obtained by benzylation of compound 6d (Scheme 2); ii)
the O-TBDMS-protected methylphosphonate ester 15; and iii) the phosphonated N-(2-
propynyl)carboxamides 18a,b, prepared from propargylamine 16 (Scheme 3).
O
O
O
O
O
Cl
P OEt
OEt
P OH
OH
NH2
HN
( )n
HN
( )n
HN
( )n
i,ii
iii
iv
1
6
For 18b
1
1
7a : n = 1
7b : n = 2
18a : n = 1
18b : n = 2
19b : n = 2
Scheme 3.
o
Reagents and conditions:- (i) triethylamine, dry THF, 0 C; (ii) chloroacetyl chloride or 3-
chloropropanoyl chloride; (iii) triethyl phosphite 6 h, 120-150 ºC; and iv) TMSBr, dry DCM,
2
N , 24 h.
While the purified phosphonic esters could be used directly as possible pro-drugs in the
P.falciparum parasite (pLDH) bioassays, hydrolysis to the corresponding acids was required
for the in vitro evaluation of Pf-DXR inhibition potential − the Pf-DXR enzyme lacking the
necessary esterases. Hydrolysis of three of the most active esters (9c, 9d and 18b) was
8
effected, using five equivalents of bromotrimethylsilane in dry DCM under nitrogen, and
afforded the corresponding phosphonic acids 10c, 10d and 19b.
6

2
.2. Pf parasite and Pf-DXR Inhibition and Toxicity Assays
Cytotoxicity (using HeLa cells) and malaria parasite lactate dehydrogenase (pLDH) assays
were first conducted using 20 µM solutions of the synthesised compounds in triplicate, and
the results are illustrated in Figure 2 and summarised in Table 2. The phosphonate esters
appear to exhibit minimal growth inhibition of the HeLa cells and, in most cases, 100% HeLa
cell viability was observed. IC50 values were then determined for compounds 5b, 5c, 18b, 14,
9
c and 9d (all of which inhibited the parasite completely at a ligand concentration of 20 µM)
and for compound 14 (which, at 20 µM, exhibited < 20% malaria parasite viability). The
percentage parasite cell viability was plotted against the logarithm of the ligand concentration
(
Figure 3) and IC50 values were determined using the trend-line. Surprisingly, the non-
benzylated compound 5b, which contains only one methylene group in the hydrophobic
patch, has the lowest IC50 value at 10.1 µM. Under the same conditions, chloroquine
exhibited an IC50 value of 0.07 µM. The remaining ligands also gave encouraging IC50 values
in the low µM range (5c: 23.1; 9c: 22.3; 9d: 15.9; 14: 16.4; 18b: 20.1).
Figure 2. Cell toxicity (HeLa cell viability) screening and anti-malarial (P.falciparum pLDH)
assay of the synthesised phosphonate esters (20 µM).
7

Table 2. Residual % P.falciparum viability and toxicity (HeLa cell viability) data
for 20 µM solutions of the synthetic ligands.
Compound
Plasmodium
falciparum
SD
HeLa
SD
Viability
%
Viability %
5
5
a
50.91
-7.26
-19.39
52.42
64.13
54.54
35.19
55.44
-5.33
-8.05
59.60
51.13
6.84
0.96
0.80
0.64
5.67
16.90
6.98
4.95
8.06
2.56
9.66
2.96
2.37
2.87
4.42
1.92
3.02
102.44
122.76
130.59
116.80
83.14
7.81
3.92
2.06
7.91
4.07
3.72
4.85
10.90
1.47
1.81
6.46
10.50
6.45
3.01
6.98
7.00
b
5
c
5
d
5
e
6d
9a
9b
119.28
120.60
109.67
112.93
120.15
118.23
104.83
107.74
85.68
9
c
9
d
9
1
1
1
e
3
4
5
61.87
47.96
-4.04
18a
103.65
91.95
18b
Compound
IC50
(µM)
5
5
b
c
10.12
23.1
1
9
9
1
8b
20.09
22.3
15.85
16.38
c
d
4
Chloroquine
~0.07108
Figure 3. The inhibitory effect of selected compounds, showing percentage viability of the
P.falciparum parasite at various concentrations and corresponding IC50 values.
8

The bioassay results reveal a number of interesting features.
i)
Six of the phosphonate esters (5b, 5c, 9c, 9d, 14 and 18b) exhibit encouraging
inhibition of the parasite and very low toxicity at a concentration of 20 µM. In
fact, five of these ligands appear to exhibit ca. 0% Pf viability at this
concentration!
ii)
Further detailed analysis of the six most active ligands reveals their IC50 values to
be in the low µmolar range (10 - 23).
iii)
Unexpectedly, introduction of the N-benzyl group coupled with the presence of a
bi-methylene bridge, aimed at enhancing PfDXR inhibition, appears to have
increased inhibitory activity against the Pf parasite in only one case; the N-
benzylated ligand 9d exhibits significantly better inhibitory activity (ca. 0% Pf
viability at 20 µM) than the non-benzylated, mono-methylene-bridged analogue
5
d (52% Pf viability at 20 µM).
iv)
Increasing the length of the hydrophobic patch between the amide and
phosphonate ester moieties (from one to two methylene groups) in the
phosphonated N-(2-propynyl)carboxamides clearly enhances inhibition activity
significantly (Pf viability: 18a: 48%; 18b: ca. 0%). A similar pattern is exhibited
by the N-benzylated analogues 5d and 9d (Pf viability: 5d: 52%; 9d: ca. 0%).
However, changing the length of the hydrophobic patch has little effect on the
significant levels of inhibitory activity exhibited by the mono-methylene-bridged
ligand 5c and the bi-methylene-bridged, N-benzylated analogue 9c − both of
which exhibit ca. 0% Pf viability at 20 µM and comparable IC50 values.
The four most active N-benzylated analogues each contain coordinating
heteroatoms in their aryl (or heteroaryl) groups [5b: Ar = 3-(hydroxymethyl)-
phenyl; 5c and 9c: Ar = 2-pyridyl; and 9d: Ar = 3-hydroxyphenyl] − reflecting,
perhaps, the importance of their hydrogen-bonding donor and/or acceptor
capacity.
v)
vi)
The evident activity of the N-propynylated derivative containing a bi-methylene
spacer 18b raises the possibility of using Click Chemistry methodology to access
more structurally diverse ligands as potential antimalarial agents.
9

The parasite is expected to possess the esterases necessary to hydrolyse the phosphonate
esters and thus release the phosphonic acids, designed as PfDXR-specific ligands. In order to
establish whether the observed Pf parasite inhibition by the most active phosphonate esters
was due to binding of their hydrolysed analogues to the PfDXR active-site, PfDXR enzyme
inhibition assays of three of the corresponding phosphonic acids were undertaken.
Surprisingly, 20 µM solutions of these compounds exhibited very little, if any, activity
against Pf DXR (10c and 10d: 0%; 19b: 6.7%) compared to the known inhibitor FR900098
(
73.3% inhibition). These results indicate that the phosphonate esters are inhibiting the Pf
parasite via a different but, as yet, unknown mechanism − a conclusion supported by the
unexpected SAR data discussed above.
In expectation that the synthetic ligands would bind to the PfDXR receptor, in silico docking
studies of selected benzylated and non-benzylated ligands were undertaken, following
9
previously reported methods but using a flexible docking approach. The PfDXR crystal
1
1
structure was accessed from the Research Collaboratory for Structural Bioinformatics
1
RCSB) protein data bank using discovery studio. The best conformation obtained in each
2
(
case appeared to correlate well with the active-site cavity of the PfDXR crystal structure; as
illustrated for the docking of phosphonic acid 10a (Figure 4), potential hydrogen-bonding
interactions can be identified while the benzyl group occupies a hydrophobic cavity and is
appropriately orientated for π-stacking with the indole moiety of a proximate tryptophan
residue. However, the bioassay data demand a different explanation for the observed activity
of the phosphonate esters reported in this study. It is thus apparent that other factors, such as,
ligand solvation and receptor hydration effects may need to be considered in future docking
studies.
3. Conclusions
Sixteen N-benzylated and non-benzylated phosphonate esters have been synthesised as
potential anti-malarial agents, six of which exhibit significant activity at low micromolar
concentrations against the Plasmodium falciparum parasite. Although designed specifically
as Pf DXR inhibitors, it is apparent from the bioassay data that a different mechanism of
action is involved.
1
0

1
1
Figure 4. Docked conformation of phosphonic acid 10a in the PfDXR active-site (3AU9).
Protein active-site residues are shown in wire frame and the ligand in stick model, all
coloured by atom type. Hydrogen–bonding interactions are shown as green dashed lines.
4
. Experimental
4
.1. General methods
NMR spectra were typically recorded on Bruker 300 or 400 MHz spectrometers in CDCl
3
and
)].
calibrated using solvent signals [δ : 7.26 ppm for residual CHCl . δ : 77.0 ppm (CDCl
H
3
C
3
Melting points were measured using a hot stage apparatus and are uncorrected. IR spectra
were recorded neat on a Perkin Elmer Spectrum 100 FT-IR spectrometer with a diamond
window. High-resolution mass spectra (HRMS) were recorded on a Waters API Q-TOF
Ultima spectrometer (University of Stellenbosch, Stellenbosch, South Africa).
1
1

4
4
.2. Synthesis
.2.1. General procedure for the reaction of primary amines with benzyl bromide.
9
,13,14
Compounds 5a-5e, 6a-6e and 7a-7e are known
but a different synthetic approach to
compounds 7a-7e, illustrated by the following example, was used in this work. Benzyl
bromide (1.90 mL, 16 mmol) was added to a mixture of 3-aminobenzyl alcohol 4b (2.0 g, 16
mmol) and NaHCO
overnight under nitrogen. Water (20 mL) was then added and the aqueous layer extracted
with CHCl (2 × 50 mL). The combined organic extracts were then washed with brine (2 × 30
mL), dried with anhydrous MgSO , filtered and concentrated in vacuo. Chromatography
PLC on silica gel; elution with hexane-EtOAc (5:1)] of the residue gave two fractions.
Fraction 1: N-Benzyl-3-(hydroxymethyl)aniline 7b as a brown oil (2.17 g, 63%); νmax/cm
385 (OH) and 3340 (NH); δ /ppm (400 MHz; CDCl ) 2.87 (1H, s, NH), 4.21 (2H, s,
NCH Ph), 4.46 (2H, s, CH OH), 6.45 (1H, d, J = 8.1 Hz, Ar-H), 6.52 (1H, s, Ar-H), 6.59
1H, d, J = 7.51 Hz, Ar-H), 7.76 (1H, t, J = 7.76 Hz, Ar-H) and 7.16-7.28 (5H, overlapping
m, Ar-H); δ /ppm (100 MHz; CDCl ) 48.3 (NCH Ph), 65.5 (CH OH), 111.4, 112.1, 116.2,
27.4, 127.6, 128.7, 129.6, 139.4, 142.2 and 148.5 (Ar-C).
Fraction 2: N-Benzyl-3-[(benzyloxy)methyl]aniline 11 as white crystals (1.08 g, 19%), m.p.
3
(1.36 g, 16 mmol) in dry THF (10 mL). The mixture was stirred at r.t.
3
4
[
-1
3
H
3
2
2
(
C
3
2
2
1
o
+
-
8
8-90 C; [HRMS: m/z calculated for C21
H
22NO (MH ) 304.1701. Found 304.1698]; νmax/cm
) 4.57 (2H, s, NCH Ph), 4.66 [4H, s, O(CH ], 6.68
1H, d, J = 7.41 Hz , Ar-H), 6.71 (1H, m, Ar-H), 6.78 (1H, br, s, NH), 7.16 (1H, m, Ar-H),
.24-7.26 (7H, overlapping m, Ar-H), 7.30-7.33 (4H, overlapping m, Ar-H); δ /ppm (100
MHz; CDCl ) 46.3 (NCH Ph), 75.1 [2C, O(CH ] 111.7, 112.5, 115.9, 126.7, 127.0, 127.5,
28.2, 128.9, 129.1, 129.7, 136.2, 140.3, 141.5 and 146.8 (Ar-C).
1
3
384 (NH), δ
H
/ppm (400 MHz; CDCl
3
2
2 2
)
(
7
C
3
2
2 2
)
1
4
.2.2. General procedure for the preparation of the 3-chloropropanamides 8a-e.
N-Benzyl-3-bromoaniline 7a (0.68 g, 2.2 mmol) was dissolved in dry THF (20 mL) and NaH
0.10 g, 4.2 mmol) was added in portions. The mixture was stirred for 30 min and 3-
chloropropanoyl chloride (0.22 mL, 2.2 mmol) was then added slowly to control the
evolution of hydrogen. After stirring overnight at r.t. under N , the solvent was removed in
vacuo, and the crude product was extracted into EtOAc (2 × 50 mL). The extracts were
combined, washed sequentially with satd. aq. NaHCO (2 × 30 mL), distilled water (2 × 30
mL) and brine (2 × 30 mL), dried (MgSO ) and filtered, and the solvent was removed in
vacuo. Chromatography of the residue [PLC on silica gel; elution with hexane-EtOAc (7:3)]
(
2
3
4
1
2

afforded N-benzyl-N-(3-bromophenyl)-3-chloropropanamide 8a as a yellow oil (0.45 g, 50%)
+
-1
[
HRMS: m/z calculated for C16
H
16NOClBr (MH ) 352.0104. Found 352.0103]; νmax/cm :
/ppm (400 MHz; CDCl ) 2.47 (2H, t, J = 6.53 Hz, CH CO), 3.74 (2H, t, J =
Cl), 4.82 (2H, s, NCH Ph), 6.83 (1H, d, J = 7.66 Hz, Ar-H), 7.10-7.19 (4H,
1
6
649 (C=O); δ
H
3
2
.64 Hz CH
2
2
overlapping m, Ar-H), 7.20 (3H, overlapping m, Ar-H), and 7.39 (1H, d, J = 8.06 Hz, Ar-H);
/ppm (100 MHz; CDCl ) 37.3 (CH CO), 40.1 (CH Cl) and 53.2 (NCH Ph), 123.1, 127.6,
27.8, 128.7, 128.9, 131.0, 131.7, 131.7, 136.8 and 143.1 (Ar-C) and 169.3 (C=O).
δ
C
3
2
2
2
1
4
.2.2.1. N-Benzyl-3-chloro-N-[3-(hydroxymethyl)phenyl]propanamide 8b as a pale yellow
+
Cl (MH ) 304.1104. Found
oil (0.27 g, 36%). [HRMS: m/z calculated for C17
H18NO
2
-
1
3
04.1107]; νmax/cm : 3346 (OH) and 1649 (C=O); δ
6.72 Hz, CH CO), 3.71 (2H, t, J = 6.73 Hz, CH Cl), 4.59 (2H, s, NCH
CH OH), 6.82 (1H, td, J = 2.14 and 4.57 Hz, Ar-H), 6.95 (1H, s, Ar-H), 7.11-7.19 (5H,
overlapping m, Ar-H) and 7.24 (2H, m, Ar-H); δ /ppm (75 MHz; CDCl ) 37.3 (CH CO),
OH), 126.7, 127.5, 127.6, 127.7, 128.6, 128.9,
H
/ppm (300 MHz; CDCl
3
) 2.47 (2H, t, J
=
2
2
2
Ph), 4.86 (2H, s,
2
C
3
2
4
1
4
2 2 2
0.3 (CH Cl), 53.3 (NCH Ph) and 64.5 (CH
29.9, 137.2, 142.0 and 143.11 (Ar-C) and 169.6 (C=O).
.2.2.2. N-Benzyl-3-chloro-N-(2-pyridyl)propanamide 8c as a yellow oil (0.28 g, 33%)
+
-1
[
HRMS: m/z calculated for C15
C=O); δ /ppm (400 MHz; CDCl
Cl), 4.51 (2H, s, NCH Ph), 6.47 (1H, d, J = 8.74 Hz, Ar-H), 6.63 (1H, t, J = 6.33 Hz,
Ar-H), 7.29-7.36 (5H, overlapping m, Ar-H), 7.54 (1H, ddd, J = 1.70, 7.19 and 8.79 Hz, Ar-
H) and 7.92 (1H, d, J = 4.95 Hz, Ar-H); δ /ppm (100 MHz; CDCl ) 39.3 (CH CO), 40.3
CH Cl) and 46.1 (NCH Ph), 118.0, 112.0, 126.9, 127.5, 128.8, 140.7, 141.7, 142.1 and
44.2 (Ar-C) and 17.4 (C=O).
.2.2.3. N-Benzyl-3-chloro-N-(3-hydroxyphenyl)propanamide 8d as a brown oil (0.33 g,
H
15ClN
2
O (MH ) 274.2738. Found 274.2735]; νmax/cm : 1661
(
H
3
) 2.82 (2H, t, J = 6.94 Hz, CH CO), 3.80 (2H, t, J = 6.94
2
Hz, CH
2
2
C
3
2
(
2
2
1
4
+
-
3
9%) {[HRMS: m/z calculated for C16
H17NO
2
Cl (MH ) 290.0948. Found 290.0941]; νmax/cm
/ppm (400 MHz; CDCl ) 2.82 (2H, t, J = 6.94 Hz, CH CO),
Cl), 4.51 (2H, s, NCH Ph), 6.47 (1H, d, J = 8.74 Hz, Ar-H),
.63 (1H, t, J = 6.33 Hz, Ar-H), 7.29-7.36 (5H, overlapping m, Ar-H), 7.54 (1H, ddd, J =
.70, 7.19 and 8.79 Hz, Ar-H) and 7.92 (1H, d, J = 4.95 Hz, Ar-H); δ /ppm (100 MHz;
) 39.3 (CH CO), 40.3 (CH Cl) and 46.1 (NCH Ph), 118.0, 112.0, 126.9, 127.5, 128.8,
40.7, 141.7, 142.1 and 144.2 (Ar-C) and 17.4 (C=O); and the bis-acylated analogue,
-(N-Benzyl-3-chloropropanamido)phenyl 3-chloropropanoate 12 as a brown oil (0.16 g,
1
:
3345 (OH) and 1668 (C=O); δ
H
3
2
3
6
1
.80 (2H, t, J = 6.94 Hz, CH
2
2
C
CDCl
3
2
2
2
1
3
1
+
-
3 2
4%) [HRMS: m/z calculated for C19H19NO Cl (MH ) 380.0820. Found 380.0823]; νmax/cm
1
3

1
:
1727 (O-C=O) and 1668 (N-C=O); δ
CH CO), 3.77 (4H, m, 2 × CH Cl), 4.66 (2H, s, NCH
6H, overlapping m, Ar-H) and 7.29 (1H, t, J = 8.15 Hz, Ar-H); δ
H 3
/ppm (400 MHz; CDCl ) 2.58-2.66 (4H, m, 2 ×
2
2
2
Ph), 6.68 (2H, m, Ar-H), 7.03-7.15
(
C
/ppm (100 Hz; CDCl
3
)
3
1
4
6.5 and 37.4 (2 × CH
2
CO), 40.5 and 42.1 (2 × CH
2
Cl) and 49.9 (NCH Ph), 113.4, 116.2,
2
18.2, 127.7, 128.6, 129.9, 130.8, 137.1, 142.9 and 153.6 (Ar-C), 170.9 and 172.0 (2 × C=O).
.2.2.4. N-Benzyl-3-chloro-N-(3-nitrophenyl)propanamide 8e as a pale yellow oil (0.48 g,
+
-
5
2%) [HRMS: m/z calculated for C16
H
16
N
2
O
3
Cl (MH ) 319.0849. Found 319.0842]; νmax/cm
) 2.47 (2H, t, J = 6.43 Hz, CH CO), 3.76 (2H, t, J
Ph), 7.09-7.12 (2H, overlapping m, Ar-H), 7.20-7.2
4H, overlapping m, Ar-H), 7.47 (1H, t, J = 8.09 Hz, Ar-H), 7.84 (1H, s, Ar-H) and 8.13 (1H,
d, J = 8.05 Hz, Ar-H); δ /ppm (75 MHz; CDCl ) 37.4 (CH CO), 40.0 (CH Cl) and 53.2
NCH Ph), 123.4, 123.8, 128.1, 128.4, 128.9, 130.7, 135.1, 136.2, 142.8 and 148.9 (Ar-C)
and 169.1 (C=O).
1
:
H
1659 (C=O); δ /ppm (300 MHz; CDCl
3
2
=
6.53 Hz, CH Cl), 4.89 (2H, s, NCH
2
2
(
C
3
2
2
(
2
4
.2.3. General procedure for Michaelis-Arbuzov reactions of N-benzyl-3-chloro-
propanamides 9a-e.
A mixture of N-benzyl-N-(3-bromophenyl)-3-chloropropanamide 7a (0.25 g, 0.72 mmol) and
o
triethyl phosphite (0.25 mL, 1.44 mmol) was heated under reflux at 120-150 C for 6 h under
2
N . The reaction was monitored by TLC and, upon completion of the reaction, the crude
product was chromatographed [PLC on silica gel; elution with EtOAc] to afford diethyl 2-[N-
benzyl-N-(3-bromophenyl)carbamoyl]ethylphosphonate 9a as a yellow oil (0.16 g, 51%)
+
-1
[
HRMS: m/z calculated for C20
H26NO
4
BrP (MH ) 454.0763. Found 454.0777]; νmax/cm :
/ppm (300 MHz; CDCl ) 1.18 (6H, t, J = 7.06 Hz, 2 × CH ),
P), 2.25 (2H, m, CH CO), 3.96 (4H, m, 2 × OCH CH ), 4.79 (2H, s,
Ph), 6.82 (1H, d, J = 7.53 Hz, Ar-H), 7.09-7.13 (4H, overlapping m, Ar-H), 7.19 (3H,
overlapping m, Ar-H) and 7.40 (1H, d, J = 7.93 Hz, Ar-H); δ /ppm (75 MHz; CDCl ) 16.5
), 20.9 (d, JP-C = 143.67 Hz, CH P), 27.9 (CH CO), 53.5
Ph), 61.8 (d, JP-C = 6.44 Hz, 2 × OCH CH ), 123.1, 127.5, 127.8, 128.7, 128.9, 131.1,
1
2
654 (C=O) and 1240 (P=O); δ
H
3
3
.03 (2H, m, CH
2
2
2
3
NCH
2
C
3
(
(
d, JP-C = 6.02 Hz, 2 × CH
3
2
2
NCH
2
2
3
1
31.2, 131.7, 136.9 and 143.2 (Ar-C) and 170.7 (C=O).
4
.2.3.1. Diethyl 2-{N-benzyl-N-{[3-(hydroxymethyl)phenyl]carbamoyl}ethylphosphonate 9b
as a yellow oil (0.15 g, 47%) following flash chromatography (on silica gel; elution with
+
P (M-1) 404.1625. Found 404.1627];
EtOAc) [HRMS: m/z calculated for C21
H27NO
5
-
1
ν
H 3
max/cm : 3345 (OH), 1664 (C=O) and 1245 (P=O); δ /ppm (400 MHz; CDCl ) 1.25 (6H, t, J
1
4

=
6.98 Hz, 2 × CH
OCH CH ), 4.65 (2H, s, NCH
.07 (1H, s, Ar-H), 6.92 (1H, d, J = 7.08 Hz, Ar-H), 7.19 (2H, d, J = 3.6 Hz), 7.24-7.29 (6H,
overlapping m, Ar-H and OH) and 7.07 (1H, d, J = 7.60 Hz, Ar-H); δ /ppm (100 MHz;
CDCl ) 16.5 (d, JP-C = 6.02 Hz, 2 × CH ), 20.4 (d, JP-C = 143.22 Hz, CH P), 29.9 (CH CO),
3.4 (NCH Ph), 61.9 (d, J= 6.44 Hz, 2 × OCH CH ), 64.6 (CH OH), 122.7, 126.7, 126.9,
3 2 2
), 2.12 (2H, m, CH P), 2.34 (2H, m,CH CO), 4.01 (4H, m, 2 ×
2
3
2 2
Ph), 4.85 (2H, s, CH OH), 6.89 (1H, d, J = 7.45 Hz, Ar-H),
7
C
3
3
2
2
5
1
4
2
2
3
2
27.0, 127.6, 127.7, 128.6, 128.9, 129.9, 131.4, 131.6 and 135.3 (Ar-C) and 163.7 (C=O).
.2.3.2. Diethyl 2-[N-benzyl-N-(2-pyridyl)carbamoyl]ethylphosphonate 9c as a yellow oil
+
P (MH ) 377.1630. Found 377.1627];
(
0.14 g, 49%) [HRMS: m/z calculated for C19
H26NO
4
-
1
ν
max/cm : 1670 (C=O) and 1248 (P=O); δ
× CH ), 2.17 (2H, m, CH P), 2.58 (2H, m, CH
s, NCH Ph), 7.22-7.31 (7H, overlapping m, Ar-H), 7.67 (1H, td, J = 1.92 and 7.77 Hz, Ar-H)
and 8.52 (1H, d, J = 3.66 Hz, Ar-H); δ /ppm (100 MHz; CDCl ) 16.5 (d, JP-C = 6.06 Hz, 2 ×
CH ), 21.7 (d, JP-C = 143.57 Hz, CH P), 28.3 (CH CO), 51.7 (NCH Ph), 61.6 (d, JP-C = 6.42
Hz, 2 × OCH CH ), 121.9, 122.5, 127.5, 127.8, 128.0, 128.1, 128.6, 135.1, 137.4, 138.6
and149.4 (Ar-C) and 155.0 (C=O).
H
/ppm (400 MHz; CDCl
3
) 1.27 (6H, t, J = 6.45 Hz,
2
3
2
2
CO), 4.06 (4H, m, 2 × OCH
2
CH ), 5.10 (2H,
3
2
C
3
3
2
2
2
2
3
4
.2.3.3. Diethyl 2-[N-benzyl-N-(3-hydroxyphenyl)carbamoyl]ethylphosphonate 9d as a
+
P (MH ) 392.1627. Found
yellow oil (0.21 g, 51%) [HRMS: m/z calculated for C20
H27NO
5
-
1
3
1
2
6
92.1623]; νmax/cm : 3340 (OH), 1667 (C=O) and 1242 (P=O); δ
.28 (6H, t, J = 6.98 Hz, 2 × CH ), 2.12 (2H, m, CH P), 2.43 (2H, m, CH
× OCH CH ), 4.86 (2H, s, NCH Ph), 6.45 (1H, d, J = 7.74 Hz, Ar-H), 6.54 (1H, s, Ar-H),
.77 (1H, dd, J = 1.68 and 8.17 Hz, Ar-H), 7.11 (1H, t, J = 8.01 Hz, Ar-H) and 7.21-7.24
5H, overlapping m, Ar-H); δ /ppm (100 MHz; CDCl ) 16.4 (d, JP-C = 6.20 Hz, 2 × CH ),
P), 27.3 (CH CO), 53.5 (NCH Ph), 62.2 (d, JP-C = 6.43 Hz, 2
), 115.5, 115.7, 119.3, 127.5, 128.5, 128.9, 130.4, 137.5, 142.8 and 158.2 (Ar-C)
and 171.1 (C=O).
H
/ppm (400 MHz; CDCl
3
)
3
2
2
CO), 4.03 (4H, m,
2
3
2
(
C
3
3
2
1.6 (d, JP-C = 143.44 Hz, CH
2
2
2
×
OCH CH
2
3
4
.2.3.4. Diethyl 2-[N-benzyl-N-(3-nitrophenyl)carbamoyl]ethylphosphonate 9e as a yellow
+
P (MH ) 421.1526. Found
oil (0.35 g, 59%) [HRMS: m/z calculated for C20
H
26
N
2
O
6
-
1
4
21.1522]; νmax/cm : 1661 (C=O) and 1243 (P=O); δ
6.98 Hz, 2 × CH ), 2.06 (2H, m, CH P), 2.25 (2H, m, CH
OCH CH ), 4.86 (2H, s, NCH Ph), 7.09-7.26 (6H, overlapping m, Ar-H), 7.46 (1H, t, J =
.04 Hz, Ar-H), 7.84 (1H, s, Ar-H) and 8.12 (1H, d, J = 7.46 Hz, Ar-H); δ /ppm (100 MHz;
CDCl ) 16.4 (d, JP-C = 6.20 Hz, 2 × CH ), 21.8 (d, JP-C = 143.8 Hz, CH P), 28.0 (CH CO),
H
/ppm (400 MHz; CDCl
3
) 1.18 (6H, t, J
=
3
2
2
CO), 3.96 (4H, m, 2 ×
2
3
2
8
C
3
3
2
2
1
5

5
1
2 2 3
3.4 (NCH Ph), 61.8 (d, JP-C = 6.42 Hz, 2 × OCH CH ), 123.3, 123.7, 128.0, 128.4, 128.8,
30.7, 135.1, 136.3, 142.9 and 148.9 (Ar-C) and 170.7 (C=O).
4
.2.4. Preparation of the O-benzylated analogues 13 and 14.
To a solution of diethyl [N-(3-hydroxyphenyl)carbamoyl]ethylphosphonate 6d (0.35 g, 1.2
mmol) dry THF (5mL), NaH (0.030 g, 1.3 mmol) was added in portions, followed by benzyl
bromide (0.19 mL, 1.5 mmol) in dry THF (10 mL). The mixture was refluxed for 4 h., and
the crude product was flash chromatographed [on silica gel; elution with hexane-EtOAc
(
3:2)] to afford two fractions.
Fraction 1: Diethyl 2-{[3-(benzyloxy)phenyl]carbamoyl}ethylphosphonate 13 as a yellow oil
+
P (MH ) 329.1627. Found 392.1616];
(
0.077 g, 16%). [HRMS: m/z calculated for C20
H27NO
5
-
1
ν
max/cm : 1647 (C=O); δ
P), 2.71 (2H, d, J = 6.19 Hz, CH
Ph), 6.69 (1H, d, J = 7.35 Hz, Ar-H), 7.11-7.48 (8H, overlapping m, Ar-H) and 9.07
1H, s, NH); δ /ppm (75 MHz; CDCl ) 16.5 (d, JP-C = 5.73 Hz, 2 × CH ), 21.1 (d, JP-C = 143.7
Hz, CH P), 30.2 (CH CO), 62.3 (d, JP-C = 6. 32 Hz, 2 × OCH CH ), 70.1 (OCH Ph), 106.5,
10.8, 112.3, 127.6, 128.0, 128.6, 128.8, 129.7, 137.2, 140.0 and 159.5 (Ar-C) and 169.4
C=O).
H
/ppm (300 MHz; CDCl
3
) 1.30 (6H, t, J = 1.31 Hz, 2 × CH
3
), 2.18
(
2H, m, CH
2
2
CO), 4.11 (4H, m, 2 × OCH
2
CH ), 5.05 (2H, s,
3
OCH
2
(
C
3
3
2
2
2
3
2
1
(
Fraction 2: Diethyl N-benzyl-{[3-(benzyloxy)phenyl]carbamoyl}ethylphosphonate 14 as a
+
P (MH ) 482.2096. Found
yellow oil (0.27 g, 46%) [HRMS: m/z calculated for C27
H33NO
5
-
1
4
82.2094]; νmax/cm : 1645 (C=O); δ
CH ), 1.98 (2H, m, CH P), 2.25 (2H, m, CH
CH Ph), 4.84 (2H, s, OCH Ph), 6.46 (2H, s, Ar-H), 6.82 (1H, d, J = 8.57 Hz, Ar-H) and 7.05-
.26 (11H, overlapping m, Ar-H); δ /ppm (75 MHz; CDCl ) 16.5 (d, JP-C = 6.01 Hz, 2 ×
CH ), 21.6 (d, JP-C = 142.7 Hz, CH P), 27.7 (CH CO), 53.4 (NCH Ph), 61.7 (d, JP-C = 6.36
Hz, 2 × OCH CH ), 70.4 (OCH Ph), 114.9, 115.2, 121.0, 127.6, 127.7, 128.3, 128.5, 128.8,
29.0, 130.5, 136.5 and 137.4 (Ar-C) and 159.8 (C=O).
H
/ppm (300 MHz; CDCl
3
) 1.13 (6H, t, J = 6.37 Hz, 2 ×
3
2
2
CO), 3.92 (4H, m, 2 × OCH
2 3
CH ), 4.73 (2H, s,
2
2
7
C
3
3
2
2
2
2
3
2
1
4
.2.5. Tert-butyldimethylsilyl ether-protected phosphonate ester 15. To a solution of diethyl
[
(
N-(3-hydroxyphenyl)carbamoyl]methylphosphonate 5d (0.50 g, 1.7 mmol) and imidazole
o
0.15 g, 2.3 mmol) in dichloromethane (20 mL) at 0 C (ice bath) was added tert-
butyldimethylsilyl chloride (0.29 g, 1.9 mmol) in portions, and the mixture was stirred at r.t.
for 2 h. The mixture was diluted with DCM (10 mL) and washed with water (2 × 20 mL) and
1
6

4
brine (2 × 20 mL). The organic layer was dried over MgSO , filtered and concentrated in
vacuo. The crude product was purified by column chromatography [on silica gel; elution with
hexane-EtOAc (9:1)] to afford the tert-butyldimethylsilyl ether-protected phosphonate ester
o
1
5 as a white solid (0.62 g, 88%), m.p. 70-72 C [HRMS: m/z calculated for C18
H
33NO
5
PSi
) 0.20
), 2.98 (2H, d, JP-H
), 6.60 (lH, dd, J =1.32 and 7.96 Hz, Ar-H),
.06 (lH, dd, J = 1.50 and 6.58 Hz, Ar-H), 7.16 (2H, m, Ar-H) and 8.71 (lH, s, NH); δ /ppm
100 MHz; CDCl ) -4.3 [Si(CH ], 16.5 (d, JP-C = 6.03 Hz, 2 × CH ), 18.3 [C(CH ], 25.8
C(CH ], 36.3 (d, JP-C = 129.4 Hz, CH P), 63.2 (d, JP-C = 6.44 Hz, 2 × OCH CH ), 112.0,
12.9, 116.3, 129.7, 138.9 and 156.3 (Ar-C) and 162.0 (C=O).
+
-1
(
[
MH ) 402.1866. Found 402.1861]; νmax/cm : 1673 (C=O); δ
6H, s, Si(CH ], 0.97 [9H, s, C(CH ], 1.35 (6H, t, J = 7.07 Hz, 2 × CH
21.0 Hz, CH P), 4.17 (4H, m, 2 × OCH CH
H 3
/ppm (400 MHz; CDCl
3
)
2
3
)
3
3
=
2
2
3
7
C
(
[
3
3
)
2
3
3 3
)
3
)
3
2
2
3
1
4
.2.6. Preparation of the acylated propargylamine derivatives 17a,b is illustrated by the
following example. A solution of propargylamine 16 (0.58 mL, 9.0 mmol) and triethylamine
o
1.3 mL, 9.0 mmol) in dry THF (20 mL) was cooled to 0 C (ice-bath) under N
(
2
. Chloroacetyl
chloride (0.72 mL, 9.0 mmol) was then added slowly through a septum to the solution which
o
was kept at 0 C for 45 min and then stirred for 2 h at r.t. The solvent was removed in vacuo
and the residue was dissolved in DCM (2×50 mL). The resulting solution was washed
sequentially with 10% dil HCl (50 mL) and water (50 mL); the aqueous layers were then re-
extracted with DCM (100 mL). The organic layers were combined, dried with anhydrous
MgSO
4
and evaporated in vacuo to afford 2-chloro-N-(2-propynyl)acetamide 17a as an off-
+
NOCl (MH )
white solid (1.13 g, 95%), m.p. 68-70 °C [HRMS: m/z calculated for C
5
H
6
-
1
1
31.0142. Found 131.0132]; νmax/cm : 3387 (NH) and 1661 (C=O); δ
CDCl ) 2.28 (1H, t, J = 2.57 Hz, CH), 4.07 (2H, s, CH Cl), 4.11 (2H, m, NCH
1H, s, NH); δ /ppm (100 MHz; CDCl ) 29.7 (CH N), 42.5 (CH Cl), 72.3 (C≡CH), 78.7
C≡CH) and 165.8 (C=O).
H
/ppm (400 MHz;
3
2
2
) and 6.83
(
(
C
3
2
2
4
.2.6.1. 3-Chloro-N-(2-propynyl)propanamide 17b (obtained using propanoyl chloride) as a
+
NOCl (MH )
white solid (1.21 g, 92%), m.p. 72-74 °C [HRMS: m/z calculated for C
6
H
8
-
1
1
46.0367. Found 146.0373]; νmax/cm : 3377 (NH) and 1653 (C=O); δ
CDCl ) 2.25 (1H, t, J = 2.55 Hz, CH), 2.66 (2H, t, J = 6.49 Hz, CH CO), 3.81 (2H, t, J = 6.49
Hz, CH Cl), 4.09 (2H, m, NCH ) and 5.83 (1H, s, NH); δ /ppm (100 MHz; CDCl ) 29.5
CH N), 39.4 (CH CO), 39.9 (CH
Cl), 72.1 (C≡CH), 79.2 (C≡CH) and 169.2 (C=O).
H
/ppm (400 MHz;
3
2
2
2
C
3
(
2
2
2
1
7

4
.2.7. Phosphonation of propargylamine derivatives 18a,b via the Arbuzov reaction is
illustrated by the following example. Triethyl phosphite (2.3 mL, 14 mmol) was added
through a septum to 2-chloro-N-(2-propynyl)acetamide 17a (0.9 g, 7 mmol) under N in an
2
oven-dried, round-bottomed flask and the resulting mixture was refluxed for 4 h. The cooled
mixture was stirred with hexane (5 × 25 mL), decanting the hexane layer each time to remove
excess triethyl phosphite. Flash chromatography [on silica gel; elution with hexane-EtOAc
(
1:4)] and evaporation of the solvent in vacuo afforded diethyl [N-(2-propynyl)carbamoyl]-
9 4
methylphosphonate 18a as a brown oil (1.08 g, 68%) [HRMS: m/z calculated for C H17NO P
+
-1
(
MH ) 234.0895. Found 234.0892]; νmax/cm : 3340 (NH), 1734 (C=O) and 1245 (P=O);
δ
H
/ppm (400 MHz; CDCl
.86 (2H, d, J = 20.58 Hz, CH
CH ) and 7.10 (1H, s, NH); δ
9.6 (CH N), 35.0 (d, JP-C = 130.9 Hz, CH
C≡CH), 79.2 (C≡CH) and 163.9 (C=O).
.2.7.1. Diethyl [N-(2-propynyl)carbamoyl]ethylphosphonate 18b (obtained from
compound 17b) as a dark brown oil (1.11 g, 65%) [HRMS: m/z calculated for C10 Cl
MH ) 248.1052. Found 248.1051]; νmax/cm : 3340 (NH), 1738 (C=O) and 1247 (P=O);
/ppm (400 MHz; CDCl ) 1.29 (6H, t, J = 7.07 Hz, 2 × CH ), 2.07 (2H, m, CH P), 2.18 (1H,
t, J = 2.56 Hz, CH), 2.49 (2H, m, CH CO), 4.01 (2H, m, CH N), 4.07 (4H, m, 2 ×
OCH CH ) and 7.13 (1H, s, NH); δ /ppm (100 MHz; CDCl ) 16.5 (d, JP-C = 6.06 Hz, 2 ×
CH ), 21.0 (d, JP-C = 143.7 Hz, CH P), 28.8 (CH CO), 29.3 (CH
OCH CH
), 71.3 (C≡CH), 79.8 (C≡CH) and 170.9 (C=O).
3
) 1.34 (6H, t, J = 7.07 Hz, 2 × CH
P), 4.05 (2H, dd, J = 2.54 and 5.34, CH
/ppm (100 MHz; CDCl ) 16.5 (d, J = 6.11 Hz, 2 × CH
P), 63.08 (d, JP-C = 6.50 Hz, 2 × OCH CH ), 71.7
3
), 2.21 (1H, t, J = 2.25 Hz, CH),
2
2
2
N), 4.16 (4H, m, 2 ×
OCH
2
3
C
3
3
),
2
2
2
2
3
(
4
H19NO
4
+
-1
(
δ
H
3
3
2
2
2
2
3
C
3
3
2
2
2
N), 61.6 (d, JP-C = 6.57 Hz, 2
×
2
3
4
4
.3. Bioassay procedures.
o
.3.1. P.falciparum pLDH assay. P. falciparum strain 3D7 parasites were cultured at 37 C
in RPMI 1640 media supplemented with 25 mM HEPES, 5% (w/v) Albumax II, 22 mM
glucose, 0.65 mM hypoxanthine, 0.05 mg/mL gentamicin and 2-4% (v/v) human erythrocytes
2 2 2
in flasks suffused with a 5% CO , 5% O , 90% N gas mixture. For compound assays,
cultures were diluted to a final concentration of 1% (v/v) erythrocytes and 2% parasitaemia
and incubated with 3-fold serial dilutions (for IC50 determination) or a 20 µM final
o
concentration of the compounds in 96-well plates (200 µL final volume) at 37 C for 48 h.
Following the incubation, residual parasite viability in individual wells was determined using
a colorimetric parasite lactate dehydrogenase (pLDH) assay – 20 sµL was removed from the
1
8

culture wells and transferred to a duplicate 96-well plate containing 125 µL per well pLDH
assay reagent (44 mM Tris buffer, pH 9, containing 0.18 M L-lactic acid, 0.13 mM
acetylpyridine adenine dinucleotide, 0.39 mM nitrotetrazolium blue chloride, 0.048 mM
phenazine ethosulphate and 0.16% (v/v) Triton X-100) and incubated at ambient temperature
for 10 – 30 minutes. Colour development was measured as absorbance at 620 nm and the
absorbance values were used to calculate percentage viability relative to control wells
containing untreated parasite cultures. IC50 values for individual compounds were determined
by non-linear regression of log (concentration) vs. percentage parasite viability plots using
GraphPad Prism (v. 5.02).
4
.3.2. DXR inhibition assay. Assays were conducted in a reaction mixture containing
1
00 mM Tris-HCl (pH 7.5), 1 mM MnCl
2
, 0.3 mM NADPH, 0.3 mM DOXP and 20 µg/ml
o
PfDXR in a total volume of 200 µL. Samples were incubated at 37 C, for 5 minutes before
the reaction was initiated by adding DOXP substrate. The decrease in absorbance at 340 nm
3
due to the decreasing concentration of NADPH (ΕNADPH = 6.3 x 10 L/mol/cm) was followed
o
®
for 10 min at 37 C using a Synergy MX (BioTek ) thermostated spectrophotometer. Reactions
were blanked against a reaction lacking the DOXP substrate. Enzyme activity in the absence
of inhibitor was deemed to be 100% (i.e. 0% inhibition) and the % inhibition, which was
determined in triplicate, was calculated relative to this. DXR activity was expressed in units
per mg of protein where 1 unit is defined as the amount of enzyme that causes oxidation of
1
µmol of NADPH per minute.
4
4
.3.3. Cell toxicity assay. HeLa cells were plated in 96-well plates to a final density 2x10
cells/well and incubated overnight in culture medium (DMEM supplemented with 10% foetal
o
incubator at 37 C.
bovine serum and penicillin/streptomycin/amphotericin B) in a CO
2
Compounds were added to the wells to a final concentration of 20 µM and incubation was
continued for 24 h. A resazurin-based toxicology reagent (Sigma-Aldrich) was added (20
µL/well) and incubated for a further 2-4 h, after which cell viability was assessed by
measuring fluorescence intensity (exc. 560 nm, em. 590 nm) in a Molecular Devices
Spectramax M3 plate reader. Percentage cell viability was calculated for individual
compound-treated wells from the fluorescence intensity readings relative to those obtained
from control wells containing untreated cells.
1
9

ACKNOWLEDGEMENTS
The authors thank the South African Medical Research Council (MRC) for a bursary
(
C.M.A.) and Rhodes University and the MRC for generous financial support. This research
project was supported by the South African Medical Research Council (MRC) with funds
from National Treasury under its Economic Competitiveness and Support Package.
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